In molecular biology, nucleic acid amplification techniques such as PCR (Polymerase Chain Reaction) are used for amplification of short polynucleotide sequences of RNA or DNA (up to 1000 nucleotides, but occasionally longer, up 10.000 nucleotides or even longer). The PCR process has been performed for the first time in 1989 by Kary Mullis.
Typically, in this process, a template of double-stranded DNA is heated to a first, denaturating temperature where DNA denatures; i.e. the double helix structure of DNA unwinds and its polynucleotide strands are separated. Usually this first temperature is 367-369 Kelvin. Depending on the sequence of the DNA template, lower temperatures or higher temperatures may be used.
In the next step, the temperature is lowered to a second, annealing temperature where primers (short, specific sequences of synthetic or non-synthetic DNA, usually 20 bases long, although primers may be longer or shorter as deemed necessary) can anneal to the denatured, single-stranded template. Usually, this second temperature is in the range of 321-343 Kelvin, more preferably 331-335 Kelvin, although higher as well as lower temperatures may be used depending on the primers used.
In the third step, the temperature is changed to a third, optimal, extension temperature, usually 345-347 Kelvin, although higher as well as lower temperatures may be used, where a polymerase enzyme, preferably a heat stable enzyme, will extend the primers with nucleotides complementary to the nucleotides of the single-stranded template.
Then the process is repeated, i.e. the mixture is returned to the denaturing temperature. This process, one calls thermal cycling. Usually 30 cycles are used to perform a PCR-reaction, although higher as well as lower cycle counts may be used.
In known embodiments (which may equally be applied in the present invention) a step-down PCR process may be applied in which the annealing temperature is lowered slightly in steps after a predetermined number of cycles.
For reactions that employ primers with high melting temperature (close to the extension temperature), a two step cycling, omitting the second, annealing temperature action may be used: annealing and extension are combined in a single step. The reaction usually takes place in a reaction vessel, called an “eppendorf tube” or in reaction plates with 96 or 384 wells. Plates and tubes are usually made of polypropylene. Other plastics may be found suitable. Other formats, such as glass tubes, are possible.
The duration of the PCR process is dependent on the speed of the reaction and on the speed and accuracy of temperature changing (thermal cycling). Over the years a number of ways to perform thermal cycling have been proposed and a number of them have been brought into practice.                The first PCR-reactions have been performed by manually changing the reaction tubes from one thermo stated water bath to the next, while timing all steps. This process was useful for the first experiments, but cumbersome and time consuming.        The first automated thermal cyclers used heating elements to heat aluminium blocks in which reaction tubes were seated. For cooling of the blocks water was used. These first machines performed PCR in approximately 4 hours.        The next generation used Peltier elements to heat and cool the blocks. These machines generate temperature transients of up to 5 K/s. Cooling is slower: at maximum −4.5 K/s. PCR can be performed in between 2 and 4 hours. Faster machines exist (Applied Bio Systems, Stratagene RoboCycler, ThermoFischer PikoCycler). The Robocycler moves plates from one temperature block to the next, using a robot arm. The Applied Bio Systems and the PikoCycler use fast temperature ramping (5 K/s and −4.5 K/s); the PikoCycler uses reaction vessels with thinner walls (average 150 μm). All of these are more or less hindered in speed by the lag in temperature of the liquid in the tubes.        Faster machines (Roche light cycler, Idaho Technology) use thermostated air to control the temperature of PCR mixtures in glass tubes. Temperature transients of 17 K/s can be reached during heating, but cooling depends on ambient temperature. PCR can be performed in 30 minutes and some cases in 20 minutes. Usually a reaction still takes approximately 1 hour.        Attempts have been made to change the temperature by creating temperature gradients inside the mixture in the test tube. Convection would then take the mixture, with its ingredients through the consecutive temperature steps automatically. This system has lately been improved by creating the temperature gradient under a slope, in order to generate better convection.        Pump systems have been designed to pump the mixture through the different temperature zones, which have been separated in space, inside a tube made from for example PTFE.        PCR on chip depends on moving very small amounts of mixture, i.e. droplets with no more than several nano liters through temperature zones, which have been created. Moving can be done by pumping or by magnetic fields, if the DNA has been labelled with magnetic beads.        Yet another system changes the temperatures in purposely-constructed cuvettes by blowing gas of the correct temperature under high pressure through the cuvette.        